IL-1ra-Fce融合基因的克隆、表达及鉴定

Cloning, Expression and Identification of IL-1ra-Fce Fusion Gene

摘要:

白细胞介素-1与免疫球蛋白E在过敏性哮喘发病中发挥着重要作用。本试验克隆了白细胞介素-1受体拮抗剂(IL-1ra)及IgE分子恒定区cDNA片段, 构建了融合基因原核表达载体IL-1ra-Fce/pBV220。将其转化大肠杆菌BL21(DE3), 实现了融合蛋白的高效表达, Western blotting结果表明表达蛋白为目的融合蛋白, 主要以包涵体形式存在; 利用分子筛和阳离子交换层析对表达产物经进行了纯化, 纯化的包涵体复性后经体外功能试验表明, 融合蛋白的活性与IL-1ra没有显著性差异; 初步药代动力学分析显示IL-1ra-Fce半衰期比IL-1ra延长了4.78倍。

Abstract:

Both interleukin-1 and IgE are important in the pathogenic mechanism of the allergy asthma. cDNA of interleukin- 1receptor antagonist (IL-1ra) and IgE were cloned and a prokaryotic expression vector IL-1ra-Fce/pBV220 was constructed. The vector was transformed into Escherichia coli i BL21(DE3). The fusion protein was expressed successfully in the form of inclusion body. The recombination protein of IL-1ra-Fce was highly purified by chromatography of gel filtration and ion exchange, which was identifited by Western blotting. The cell assay showed that the activity of IL-1ra-Fce was as high as IL-1ra in vitro after refolding. The pharmacokenetic profile of IL-1ra-Fcε and IL-1ra was analyzed, and the half time of IL-1ra-Fce is 4.78 times than that of IL-1ra.